Gastroprotective effects of both aqueous and ethanolic extracts of Lemon verbena leaves against indomethacin-induced gastric ulcer in rats

Objective(s): Regarding Lemon verbena gastroprotective effects, we investigated the protective effects of Lemon verbena extracts on reducing gastric ulcer induced by indomethacin. Materials and Methods: Rats received aqueous and ethanolic extracts of Lemon verbena (50, 100, and 200 mg/kg), zileuton (100 mg/kg), montelukast (10 mg/kg), or 1% Tween 80 in presence or absence of indomethacin (100 mg/kg). Results: Indomethacin produced stomach ulcer and increased neutrophils percentage and MDA level compared with the control group (P<0.001). Co-administration of indomethacin and zileuton, montelukast and ethanolic (200 mg/kg) (P<0.001), aqueous extract (200 mg/kg) (P<0.05) reduced ulcer compared with the indomethacin group (P<0.001). Ethanolic extracts (100 and 200 mg/kg) and aqueous extract (200 mg/kg) reduced the MDA level (P<0.001). Ethanolic (50, 100, and 200 mg/kg) and aqueous extracts (200 mg/kg) significantly decreased neutrophils percentage compared with the indomethacin group (P<0.001). Conclusion: Aqueous and particularly ethanolic extracts of Lemon verbena have protective effects on indomethacin-induced gastric ulcers.

The main mechanism for NSAIDs damage to gastroduodenal mucosa involves systemic prevention of expressed cyclooxygenase 1 (COX-1)-derived prostaglandins (6,7). COX inhibition by NSAIDs enhances the synthesis of leukotrienes (LTs) that occur by shunting the arachidonic acid metabolism towards the 5-lipoxygenase (5-LOX) pathway. Cysteinyl leukotrienes (CysLTs), such as LTC4, LTD4, and LTE4 are the main proinflammatory lipid mediators and metabolites of the 5-LOX pathway (8). LTB4 is an important factor in several neutrophile activities like adherence and chemotaxis (9). Additionally, LTs are involved in gastric mucosal complications by triggering tissue ischemia and inflammation (10). The other mechanisms include the generation of reactive oxygen species (ROS) and nitric oxide (NO), the beginning of lipid peroxidation, and penetration of neutrophils secondary to the generation of inflammatory factors. Indomethacin as a beneficial strong NSAID has been considered a drug of choice to induce gastric ulcers (11,12). Indomethacin changes the arachidonic acid metabolism in neutrophils, through inhibition of COX pathways which directs this metabolism to the 5-LOX pathway, leading to an increase in LT levels (13,14). This drug through a neutrophil-dependent process, accumulation and neutrophil adhesion, and inducible nitric oxide synthase (iNOS) as a producer of NO, initiates and advances gastric damages (15)(16)(17).
Therefore, this study aimed to evaluate the gastroprotective effect of both aqueous and ethanolic

Gastric ulcer induction
A total of 54 rats received a single dose of 100 mg/ kg indomethacin (dissolved in 0.1% tween 80) for half an hour after protective agent administrations in the protective groups and 4 hr after lack of access to food in the groups that only received indomethacin (14).

Determination of macroscopic gastric ulcer score
On the experiment day, the rats did not receive water and food for 4 hr. Extracts and control were administered intraperitoneally to rats half an hour before the oral administration of indomethacin. Four hours after the administrations of indomethacin, rats were killed and gastric tissue was removed and cut off from the posterior wall and washed in normal saline to remove the contents. Gastric ulcer index according to the severity of stomach damage was scored as below: 1= Petechia; 5= lesion size 1-3 mm; 10= lesion size up to 3 mm Finally, each stomach was placed in a separate tube and kept at the temperature of -80 °C for other experiments (14,30).

Measurement of lipid peroxidation indices (Malondialdehyde (MDA) level)
First, 10% homogenate of the stomach tissue was mixed with 1.15% potassium chloride in a container of ice, and then 0.5 ml of it mixed with 3 ml of phosphoric acid 1% and 1 ml of Thiobarbituric acid (TBA) 0.6 % in some tubes. The tubes were placed in boiling water for 45 min, and after cooling, 4 ml of n-butanol was added to each tube to remove the colored complex, and after that, they were vortexed for 1 min. Finally, the tubes were placed inside the centrifuge at 4000 rpm for 20 min at 4 °C . After completion of centrifugation, the supernatant was separated and the absorbance was read with a spectrophotometer (Jenway 6105 UV/vis, England) at 532 nm. The standard curve was drawn up in a concentration range of 0-100 nmol/ml for MDA and the concentrations were calculated and reported as nmol/g tissue value (31).

Calculating the percentage of neutrophils in the blood
Sixty minutes after administration of indomethacin, blood samples were collected from rat eyes and transferred to heparin vials and sent to the laboratory to determine neutrophil counts (14,30).

Statistical Analysis
Statistical analysis was carried out using the Prism 6 software package and the tests including one-way ANOVA test and post hoc Tukey-Kramer test were used for comparison of MDA levels and percentage of neutrophils in the blood. Moreover, the scores of gastric ulcers with Kruskal-Wallis test were evaluated and the results were shown via the median. Data were shown as mean values±standard deviation (SD). The statistical differences are significant if the P-value ≤ 0.05.

Gastric ulcer index
The gastric ulcer index was determined by evaluating the ulcer score on the stomach mucosal wall. Indomethacin administration considerably increased the ulcer index and pathological score values in the stomach tissue compared with the control group (P<0.001).The ethanolic extract of Lemon verbena (200 mg/kg), zileuton, and montelukast significantly reduced the ulcer index (P<0.001). The aqueous extract of Lemon verbena (200 mg/kg) also resulted in a substantial reduction in the ulcer index compared with the indomethacin group (P<0.05). A comparison of the protective effect of high doses of aqueous and ethanolic extracts of Lemon verbena with zileuton and montelukast did not show any significant difference. The macroscopic observations and scores of the gastric ulcers were shown in Figures 1-3.

Effect of indomethacin and extracts of Lemon verbena on lipid peroxidation induced by indomethacin in gastric tissue
In the gastric tissue, the MDA level in the indomethacin treatment group was remarkably higher when compared with the control group (P<0.001). Meanwhile, Administration of NSAIDs, especially indomethacin leads to microscopic and macroscopic wounds in animals that resemble the gastric ulcers in humans (32). In our study similar to previous studies oral administration of indomethacin at a dose of 100 mg/kg induced an obvious macroscopic gastric ulcers in rats (14,33). Inhibiting the COX enzyme by NSAIDs leading to a decrease in mucosal synthesis and bicarbonate secretion, and an increase in leukocyte accumulation is the main cause of gastric ulcers (14). Several studies revealed indomethacin, through the prevention of PGs synthesis in the arachidonic acid metabolism pathway, stimulates the 5-LOX enzyme leading to elevated LT's amounts (13,34). On the other hand, LTB (4) 12-HD/ PGR is a crucial enzyme for eicosanoid inactivation and acts an important part in the metabolism of PGs E and F, and LTB4. This enzyme is a potent chemoattractant agent for inflammatory leukocytes and 15-oxo-lipoxin A (4) (15-oxo-LXA (4)). Some NSAIDs like indomethacin apart from inhibition of COX can act as LTB (4) 12-HD/ PGR inhibitor (35,36) and thus cause an increase in LT's levels. Some studies have found that LTB4, LTC4, or LTD4 are the main factors in the development of vascular damage and mucosal lesions in the gastric tissue (37)(38)(39). One attempt has been made to investigate the protective impact of 5-LOX inhibitors and LT antagonists on the development of gastric ulcers after indomethacin intake. The results of the study highlighted that overproduction of metabolites of the 5-LOX pathway plays an important role in the development of gastric injury, and 5-LOX inhibitors and LTs antagonists' agents reverse these gastric damages induced by indomethacin (40). The most important sources of LTs are neutrophils, which are more effective in the pathogenesis of gastrointestinal tract ulcers induced by NSAIDs, especially indomethacin (30). For this reason, we carried out an experiment to investigate the effect of indomethacin on neutrophil counts. The main functions of LTs are the invitation of neutrophils and chemokines and causing neutrophil adhesion to epithelial cells. Moreover, LTs activate neutrophils to release mediators, ultimately lending to degranulation (41). Activated neutrophils exhibited microvascular disturbance and then resulted in gastric erosions following NSAIDs intake (42). Elevated LTB4 concentrations trigger chemotaxis, adhesion, and degranulation of neutrophils. These processes stimulate gastric mucosal injuries (43). The previous studies have proven that gastrointestinal ulceration resulting from indomethacin intake are due to increased LTs production followed by an increment in neutrophil infiltration and gastric MPO activity (14,41,44). Singh et.al in 2005 (30), expressed that pretreatment of leukoflone as an inhibitor of COX 1/2-5-lipoxygenase created gastroprotective functions against high amounts of produced LB4 following great infiltration of neutrophils in both rat and mice blood caused by indomethacin. Our study showed that both extracts of Lemon verbena, similar to montelukast and zileuton, dramatically reduced the gastric lesion score; inhibition of the 5-LOX pathway by these protective agents proved the feasible role of LTs in gastric damage caused by indomethacin. In a study, montelukast and curcumin (inhibitor of COX and LOX) protected the gastric tract against the damages caused by indomethacin. Besides, montelukast and curcumin reduced neutrophil adherence by a reduction in the production of LTs compared with the indomethacin group (14). Dengiz GO et al. (45) revealed montelukast through amelioration of oxidative damage and MPO function can play a gastroprotective role against indomethacin-induced gastric ulcers. The other investigation showed that montelukast is capable of decreasing the MPO function in neutrophils, prevention of LTs synthesis, and stomach mucosal permeability to involved ions and local increment in the PGs production after gastric ulcers induced by indomethacin. Furthermore, the lipid peroxidation process influences gastric ulcers through MDA production (46), hence the high levels of MDA indicate an increase in the levels of free radicals (47). It is supported by the results of the study, that the administration of indomethacin to rats resulted in a marked increase in MDA levels compared with controls.
Lemon verbena owing to having polyphenol compounds such as verbascoside and flavones exhibit strong antioxidant activity and lipoxygenase inhibition (24,48). Verbascoside is the main water-soluble phenylpropanoid glycoside component of L. citriodora extracts (24,49). The verbascoside in equimolar mixtures is a potent inhibitor of oxidative stress and because of catechol groups is capable of scavenger activity and also with sugar residue shows solubility and reducing effects (50,51). A study (48), after evaluation of various extraction procedures reported that ethanolic extract of Lemon verbena exhibited a higher capacity of recovery of organic compounds in comparison with the aqueous extract of Lemon verbena. Notably, the ethanolic extract of Lemon verbena can purify verbascoside and its analogs.
It appears that the present study confirms previous findings and contributes additional evidence that suggests ethanolic extracts of Lemon verbena due to higher recovery of verbascoside, as the main ant potent compound of lemon verbena, highlighted more gastroprotection than the aqueous extract of Lemon verbena against indomethacin-induced gastric ulcer.
Daily administration of Lemon verbena extract (water: methanol 1:1, containing 10% verbascoside) to athletes leads to a significant decrease in the levels of MDA in neutrophils and protected these cells against oxidative stress lesion compared with the control group (52). Furthermore, the aqueous extract of L. citriodora with an unknown mechanism more strongly healed gastric ulcers induced by ethanol in rats in comparison with ranitidine (53). L. citriodora owing to verbascoside compound included pharmacological activity showing anti-inflammatory activity by prevention of MPO synthesis in neutrophils and antioxidant defense against free radical productions in all different blood cell types (23). A study (27), revealed that Lemon verbena extract, because of containing verbascoside as a strong radical scavenger, exhibited potent antioxidant activity such as decrement in MDA levels. A study introduced verbascoside as a noteworthy inhibitor of both 5-LOX and protein kinase C (PKC) apart from capability in free radical scavenging and antioxidant characteristics (54). Verbascoside possessed anti-inflammatory properties as a result of a reduction in superoxide radical generation and GPx function, and consequently decrease in iNOS activity (55).
To sum up, this experiment substantiates strong gastroprotection of Lemon verbena against indomethacin-induced gastric ulcers with comparable efficacy to CysLT inhibitors such as zileuton and montelukast. The prospective usage of Lemon verbena as a protective compound versus gastric ulcer remains an open area for future research.

Conclusion
In brief, this is the first study to evaluate the protective effects of Lemon verbena against the indomethacininduced gastric ulcers in rats. The results of this study showed that intraperitoneal administration of aqueous and ethanolic extracts of Lemon verbena at a dose of 200 mg/kg has a protective effect on the development of gastric ulcers induced by indomethacin possibly by inhibiting the LOX enzyme or inhibiting LTs receptors and lipid peroxidation. Concerning the evidence on protective effects of the extracts of Lemon verbena in reducing the gastrointestinal damage induced by indomethacin, future experiments are needed to investigate the protective effects of the active ingredients of Lemon verbena on healing the gastric ulcers and to explore the involved mechanisms regarding protective effects of Lemon verbena against gastrointestinal damage caused by indomethacin.